Agronomic Approach to Iron Biofortification in Chickpea

Abstract

Chickpea (Cicer arietinum L.) is a staple crop in many developing countries where iron (Fe) deficiency is severe. The biofortification of chickpea is a possible solution to address the Fe deficiency problem. A chickpea biofortification experiment was conducted under field conditions to evaluate the effects of different doses of Fe fertilizer (0 kg ha⁻¹,10 kg ha⁻¹ and 30 kg ha⁻¹ of Fe-EDDHA) on the Fe content in seeds from 18 chickpea cultivars. The experiment was designed as a factorial combination of 18 chickpea cultivars and three Fe fertilizer doses in a randomized complete block design with four replications at two locations in Saskatchewan in 2015 and 2016. The Fe concentration in seeds across 18 different chickpea cultivars increased with Fe fertilization. Fe concentration in X05TH20-2 and CDC Frontier cultivars increased from 57 µg g⁻¹ to 59 µg g⁻¹ and from 56 µg g⁻¹ to 58 µg g⁻¹, respectively, after adding Fe fertilizer in both locations in 2015 and 2016. The biofortified seeds of these two cultivars can provide approximately 6 mg Fe 100⁻¹ g seeds. Thus, 67 and 150 g of Fe biofortified chickpea seeds can provide 50% of the recommended dietary allowance of Fe for men and women.

1. Introduction

Fe is a key micronutrient for most living organisms to conduct ubiquitous metabolic processes involving electron transfer. Examples of such metabolic processes include DNA synthesis, oxygen transport, cellular respiration and photosynthesis. Moreover, Fe is vital as a co-factor in numerous heme complexes, for example, hemoglobin, catalase and DNA helicases [1,2,3]. Although Fe in many arable lands is relatively abundant (concentration range of 20–40 g kg⁻¹), the low amount of the available form has resulted in an Fe deficiency that limits plant growth [4]. Since Fe is highly reactive to oxygen, the formation of insoluble oxidized Fe (III) restricts Fe uptake by roots, especially in high-pH and high-HCO₃⁻, calcareous soils. In high-pH and well-aerated soils, the total conc. of Fe in the soil solution was around 10⁻¹⁰ M, which is 10⁻⁴–10⁻⁵-fold less than the required amount for optimum plant growth [5,6]. In terms of Fe uptake from soil, plants are divided into two categories: Strategy I, nongraminaceous plants, and Strategy II, graminaceous plants [6]. Strategy I plant species acquire Fe after the reduction of Fe (III) chelates at the root surface, followed by the absorption of Fe (II) ions throughout the plasma membrane [7]. Since one-third of the world’s arable land is too alkaline for optimum plant growth, many studies have focused on how plants acclimatized to Fe deficiency [8]. In addition, a limited uptake of Fe was mostly observed in Strategy I plant species that depend on ferric reductase for transferring Fe [3,9]. Consequently, this condition decreased plant productivity and led to low quality, including a low Fe content in seeds that ultimately results in a public health problem. The Fe in plant-based diets is a non-heme Fe that is less bioavailable than heme Fe. As such, the prevalence of Fe-deficiency-induced anemia often occurred in populations in which the total calorie intake came from monotonous plant-based diets [1].

Biofortification is a long-term food-based approach to alleviating micronutrient deficiency. It is a strategy of producing staple food crops with increasing concentrations of bioavailable micronutrients in the edible parts that is considered more sustainable and economical [10,11]. To increase bioavailable Fe in seeds, an Fe biofortification strategy can be implemented that includes agronomy, plant breeding and genetic engineering approaches [12]. An agronomic approach could be a rapid solution to boost the Fe content in plants. Furthermore, agronomic biofortification can be integrated with other breeding-based biofortification methods [13]. Agronomic biofortification, which is also known as ferti-fortification, involves the application of fertilizer either to soil and/or to foliage to increase the bioavailability of nutrients in the edible parts of a plant [14,15]. Adding micronutrients to soil is a functional strategy to enhance the nutritional status of a plant [16,17,18]. However, there are several factors that control the increase in bioavailable nutrients in seeds. For example, the source of the fertilizer, the time and method of applying the fertilizer, and the quantity of fertilizer [19,20]. As Fe can be rapidly converted into an unavailable form when it is applied to high-pH soil, the application of an inorganic fertilizer such as ferrous sulphate (FeSO₄) could be ineffective [3]. Synthetic Fe chelates, in which Fe is combined with an organic chemical to form a chelate, create a form of Fe that is accessible to plants. Moreover, Fe chelates are soluble for a longer period than inorganic Fe. In this context, the application of chelated Fe fertilizers, for example, Fe-DTPA, Fe-EDTA and Fe-EDDHA, can be effective in high-pH soils. Among the three, Fe-EDDHA is the most effective Fe fertilizer compared to others [21].

Chickpea (Cicer arietinum L.) is a staple food crop in many African and Asian countries in which the incidence of Fe deficiency is common [1,22,23]. Based on global production, chickpea is the second most important pulse after common bean [24]. Globally, chickpea production has increased mostly gradually (96%) in developing countries [24]. Like production, the highest chickpea consumption rates occurred in South Asia and the Middle East–North Africa at 4.25 kg person⁻¹ and 2.11 kg person⁻¹ year⁻¹, respectively [25]. The growth of chickpea consumption has also increased in developed countries. In USA, the consumption of chickpea has increased almost double from 199.6 g in 2010 to 322.1 g per person per year in 2014 [26]. Chickpea is consumed in a variety of ways, for example, the green pods, immature seeds and young leaves are consumed as vegetables, whereas as the primary commodity, chickpea, is consumed as dried mature seeds in a whole, hulled or flour form [27,28]. Nutritionally, chickpea is a rich source of protein (20–22%) along with micronutrients including Fe (3.0–14.3 mg 100 g⁻¹) [29,30]. Therefore, the Fe biofortification of chickpea to produce seeds with an increased Fe concentration can mitigate Fe deficiency in populations with poor Fe intake [22]. Several studies have reported that an agronomic approach could result in higher micronutrients in the edible parts of different crops. Examples of successful agronomic biofortification include zinc (Zn) fertilizer for wheat and selenium (Se) for maize [31,32]. Moreover, in chickpea, the soil application of Zn fertilizer increased the grain Zn content and Zn yield compared to a control [33]. In addition, the foliar application of Zn and Fe fertilizer also increased the grain Zn yield in chickpea and the Fe content in the leaves, stems and grains of mung bean [33,34]. The foliar application of Se fertilizer increased the Se concentration in pea and common bean seeds [35,36]. In addition, Fe and Zn concentrations in grains of cowpea increased after applying Zn-EDTA in potting compost [37]. The combined application of Zn-DTPA and ZnSO₄ also increased Fe and Zn contents in bean under a hydroponic system [38]. Agronomic biofortification with Fe and Zn in chickpea increased yield and improved nutritional quality [39]. Fe biofortification in cowpea also showed that the combined application of ferrous sulfate and ferrous chelate in potting compost increased the Fe content of cowpea seeds compared to a control [40]. In terms of bioavailability, several authors reported that Zn and Se fertilization increased their bioavailability in human diets [41,42,43]. A previous study reported that an increase in the Fe concentration in chickpea seeds also increased Fe bioavailability [44]. The application of a bio-fertilization treatment in chickpea was the most effective at increasing seed yield [45].

Although there are few reports in chickpea biofortification with Fe and zinc (Zn), there are no reports on the agronomic approach for Fe biofortification in chickpea. Given the importance of soil Fe concentration, soil pH and HCO₃⁻ level, we hypothesised that the application of Fe fertilizer through soil increases the Fe concentration in seeds. The main objectives of this study were (1) to evaluate the effects of soil-applied Fe fertilizer on the Fe concentration in seeds; and (2) to determine a correlation between the Fe concentration and yield.

2. Materials and Methods

2.1. Description of Location and Year

This study was conducted at two locations, Elrose and Moose Jaw in Saskatchewan, Canada, during the growing seasons from May to September in 2015 and 2016. The geographical coordinates of Elrose and Moose Jaw are 51.2006° N, 108.0329° W and 50.3916° N, 105.5349° W, respectively. The soil texture at both locations is clay loam to clay. The soil climatic zones of Elrose and Moose Jaw are dark brown and brown soil zones, respectively.

2.2. Soil Sample Analysis

A soil sample analysis was conducted to examine the physico-chemical characteristics along with the concentrations of several micronutrients including Fe. Soils from both locations are calcareous (pH > 7.0), and the Fe status was above the marginal level (Figure 1).

Ten to twelve soil samples were collected diagonally from two layers (0–6″ and 6–12″) at each location for physico-chemical analyses before and after Fe fertilization. The samples were air-dried at 35 °C for five days, and a wooden roller was used to grind the soils. The ground soil samples were analyzed for different nutrients and chemical properties. A summary of soil properties prior to fertilization and after fertilization in 2015 and 2016 is shown in Table 1.

2.3. Plant Materials

Eighteen chickpea cultivars and advanced breeding lines were used in this experiment (Table 2). All the cultivars were obtained from the chickpea breeding program at the Crop Development Centre, University of Saskatchewan.

2.4. Fe Fertilizer and Application

Synthetic Fe (III) chelates (Fe-EDDHA) were used as a soil-applied fertilizer as a chelating Fe fertilizer is more effective and stable in high-pH soil (Table 3).

Eighteen chickpea cultivars (Table 2) and three rates of Fe fertilizer (Fe-EDDHA) were used in the experiment. Fe fertilizer was applied at three rates, S0 (control: no Fe application), S1 (10 kg ha⁻¹) and S2 (30 kg ha⁻¹), of Fe (Fe-EDDHA) solution with 6% actual Fe and 38.7% EDDHA. The application of Fe fertilizer was carried out via the spraying method, using a nozzle sprayer to spray the soil immediately after planting. Each low-dose plot (S1) was supplied with 9 g of the Fe-EDDHA solution, whereas the high-dose plot (S2) received 27 g. The range of the solution prepared for regular agricultural practice was from 0.05 to 0.1 g/mL of H₂O. The Fe-EDDHA solution containing 38.7% EDDHA was prepared with 0.07 g of Fe-EDDHA per ml of H₂O. By this protocol, 128 mL and 385 mL of the Fe-EDDHA solution were sprayed on the S1 and S2 plots, respectively. The time of spraying in each row of the S1 plot was 6 s, whereas in each S2 plot, it was 18 s.

2.5. Experimental Design

To evaluate the interaction effects of different doses of Fe fertilizer and chickpea cultivars, the experiment was arranged as an 18 × 3 factorial randomized complete block design (RCBD) with four replications for each location and year. Each experimental plot was 4.2 m², and the total number of plots was 216 for each location and year. The plot consisted of 3 rows with 0.31 m intra-row spacing. The seeding rate was 180 seeds per plot. The experiment was seeded using a plot seeder on 29 April and 1 May 2015 at Elrose and Moose Jaw, respectively, and on 3 May and 29 April 2016 in Elrose and Moose Jaw, respectively. General crop management practices were carried out, following the recommendation for chickpea crop in the area. No herbicide was used to manage weeds. When required, weeding at the experimental site was performed manually. In 2016, due to wet conditions in the second half of the growing season, both locations were infected with the ascochyta blight disease.

2.6. Data Collection

2.6.1. Agronomic Traits

Data were taken for the following agronomic characteristics: germination (%), node number, days to flowering, days to maturity, plant height (cm), disease score, 100-seed weight and seed yield (converted to kg ha⁻¹). Germination % was determined by counting the total plant number at each plot and was then converted into %. Node number and plant height were recorded by randomly selecting five plants from the middle row of each plot. Days to flowering was calculated from the seeding date until 50% of the plants within a plot had flowered. Like days to flowering, days to maturity was counted when 50% of the plants had changed color. At maturity, five individual plants from each plot were taken randomly to determine the biomass (dry weight). The reaction to ascochyta blight disease was scored at late podding stage (before maturity) by using a 0–9 scale [47]. A detailed rating scale for ascochyta blight on chickpea is given in Table 4.

The 100seed weight was determined by randomly taking 200-seeds that were counted using seed counter, followed by weighing with a digital balance. The weight of 200-seeds was then converted into the 100seed weight. The seed yield of each plot was recorded in grams per plot and then converted into kg ha⁻¹. The Fe yield was calculated by multiplying the seed Fe concentration by the seed yield and then converted into g ha⁻¹.

2.6.2. Seed Fe Analysis

Fe concentrations (µg g⁻¹) in mature seeds of eighteen different cultivars with three doses of Fe fertilizer were measured via flame atomic absorption spectrometry (F-AAS, Nova 300, Analytik Jena AG, Jena, Germany) at the analytical laboratory at the Department of Plant Sciences, University of Saskatchewan. Before the Fe concentration analysis, a Vulcan digester (Vulcan 84, Questron Technology, Ontario, CA, USA) was used to digest the samples. The procedures for digestion and the Fe concentration analysis were described previously [30,48]. After harvest, mature seeds were cleaned to remove any soil particles using air pressure. Cleaned seeds were ground using a cyclone sample mill (UDY Corporation, Fort Collins, CO, USA). One-half gram of powdered sample was used for determining the seed Fe concentration with three replications.

2.7. Statistical Analysis

A statistical analysis was performed following the PROC MIXED procedure of SAS version 8.0 (SAS institute Inc., Cary, NC, USA) for a factorial randomized complete block design. Initially, the analysis was conducted for each location and year, followed by a combined analysis across locations and years. The location and year and their interactions were considered random effects, whereas the effect of the cultivar and Fe fertilizer dose were considered fixed. The Least Significant Difference (LSD) test was used to separate mean values.

3. Results

The effects of the soil-applied Fe fertilizer across locations and years and their interaction (location × year) and across cultivars and doses and their interaction (cultivar*dose), along with all the two-, three- and four-way interactions on different parameters of chickpea cultivars, are presented in Table 5.

Across the sixteen different factors, cultivar (CUL) and the interaction of cultivar and location (CUL*LOC) were highly significant (p ≤ 0.01) for all parameters. Furthermore, the effect of location (LOC), year (YEAR) and replication (REP) and the interactions of location and year (LOC*YEAR), location and cultivar (LOC*CUL) and location, year and cultivar (LOC*YEAR*CUL) were also significant on most of the parameters. However, the effects of Fe fertilizer were significant only on plant biomass and seed Fe concentration. The interaction effects between cultivar and fertilizer dose (CUL*DOS), dose and year (DOS*YEAR), location, year and dose (LOC*YEAR*DOS), cultivar, year and dose (CUL*YEAR*DOS), cultivar, location and dose (CUL*LOC*DOS) and location, year, cultivar and dose (LOC*YEAR*CUL*DOS) on most of the parameters were not significant. However, the interaction effects of location and dose (LOC*DOS) were significant on most of the parameters except germination, node number, plant height and seed Fe concentration (Table 5).

The effects of soil-applied Fe fertilizer across locations, cultivars, doses and their interactions (location*cultivar), (location*dose), (cultivar*dose) and (location*cultivar*dose) on the disease ascochyta blight scores of chickpea cultivars in 2016 are presented in Table 6.

Across the eight different factors, location (LOC), cultivar (CUL), replication (REP) and the interaction of location and dose (LOC*DOS) were highly significant (p ≤ 0.01) for the ascochyta blight disease score. However, the effect of Fe fertilizer on the disease severity was not significant. The interaction effects between location and cultivar (LOC*CUL), cultivar and dose (CUL*DOS) and cultivar, location and dose (CUL*LOC*DOS) on disease were also not significant (Table 6).

3.1. Biomass

The biomass data were based on the mean dry weight of five randomly harvested plants per plot. The mean biomass (g) of each of the eighteen chickpea cultivars with three Fe fertilizer doses (0 kg ha⁻¹, 10 kg ha⁻¹ and 30 kg ha⁻¹) at both locations in 2015 and 2016 are presented in Table 7.

The effects of locations, cultivars and their interactions on biomass were significant (Table 5). Moreover, the main effect of Fe fertilizer on biomass was highly significant (p ≤ 0.01). The highest biomass was obtained from the cultivar CDC Frontier (196 g per plant), followed by CDC Cory (194 g per plant), whereas the lowest was found for CDC Alma (128 g per plant). The highest mean biomass (268 g) was obtained with 30 kg ha⁻¹ of Fe fertilizer at Elrose in 2015. The biomass of the cultivars grown with no Fe fertilizer was significantly lower than the other two doses. However, some cultivars at Moose Jaw in 2015 obtained the highest biomass with no Fe fertilizer compared to the other two doses. For instance, the cultivar CDC Luna had the highest biomass (102 g per plant) at 0 kg ha⁻¹ of Fe fertilizer compared to the other two doses (Table 7).

3.2. Seed Fe

The mean seed Fe concentrations (µg g⁻¹) of eighteen cultivars with three Fe fertilizer doses (0 kg ha⁻¹, 10 kg ha⁻¹ and 30 kg ha⁻¹) at both locations in 2015 and 2016 are presented in Table 8.

The main effects of locations, years and cultivars as well as their interactions were highly significant (p ≤ 0.01) on seed Fe concentrations (Table 5). Moreover, significant differences were found among doses. Seed Fe concentrations obtained from Elrose in the years 2015 and 2016 were significantly higher than from Moose Jaw. The highest Fe concentration in seeds was observed for cultivar X05TH20-2 (58 µg g⁻¹), followed by CDC Frontier (56 µg g⁻¹). The highest mean dose (67 µg g⁻¹) in seed Fe concentration was obtained at 30 kg ha⁻¹ of Fe fertilizer at Elrose in 2016 compared to all other doses at both locations in 2015 and at Moose Jaw in 2016. The seed Fe concentrations obtained from the no-Fe fertilizer were the lowest compared to the other two doses except for cultivar X05TH47-3 at Elrose in 2016. The lowest mean dose (40 µg g⁻¹) in seed Fe concentration was observed under the no-Fe-fertilizer treatment at Moose Jaw in 2015 compared to both locations in 2016 and Elrose in 2015 with other two doses (Table 8).

3.3. Fe Yield

The mean Fe yields (g ha⁻¹) of eighteen chickpea cultivars with three Fe fertilizer doses (0 kg ha⁻¹, 10 kg ha⁻¹ and 30 kg ha⁻¹) at both locations in 2015 and 2016 are presented in Table 9.

There were significant differences in Fe yield among cultivars and doses. The Fe yield obtained from Elrose in 2015 was significantly higher than other locations and years. The highest cultivar mean Fe yield was obtained from CDC Consul (238 g ha⁻¹), followed by CDC Corinne (230 g ha⁻¹). Furthermore, Fe fertilizers with doses of 10 kg ha⁻¹ and 30 kg ha⁻¹ yielded the highest Fe yields of 355 g ha⁻¹ and 354 g ha⁻¹, respectively, at Elrose in 2015 compared to the Fe yield at both locations in 2016 and at Moose Jaw in 2015. The Fe yield obtained from plants with no application of Fe fertilizer was lower than the other two doses for most cultivars. However, some cultivars were able to produce the highest Fe yield with a low dose (0 kg ha⁻¹) of Fe fertilizer compared to the other two doses. For instance, cultivar 1173-1 had the highest Fe yield (129 g ha⁻¹) at 0 kg ha⁻¹ of Fe fertilizer (Table 9).

4. Discussion

The application of synthetic Fe chelates on chickpeas across two years and two locations in Saskatchewan had significant effects (p ≤ 0.01) on biomass and seed Fe concentration (Table 5). The chelated Fe fertilizer at 10 kg ha⁻¹ and 30 kg ha⁻¹ improved the seed Fe concentration across cultivars and environments. In 2015 and 2016, Elrose yielded higher levels of seed Fe concentration compared to Moose Jaw. Moreover, the highest seed Fe concentration (58.5 µg g⁻¹) was obtained from CDC Frontier with a 30 kg ha⁻¹ dose of chelated Fe fertilizer, whereas the lowest (44.5 µg g⁻¹) was obtained from CDC Vanguard with no fertilizer application. At 30 kg ha⁻¹ Fe fertilizer, the seed Fe concentrations of CDC Frontier at Elrose and Moose Jaw in 2016 increased by 15% and 16%, respectively, compared to the control. However, the seed Fe concentrations at different doses across environments only gained 5–11% increases compared to the control. This suggests that the application of chelated Fe, which is the dominant form of Fe in alkaline soil, provided readily available Fe to the roots of the chickpea plants. Consequently, the Fe concentration in seeds increased compared to the control. Our findings are in agreement with the findings of Moraghan et al. [49], who reported that application of Fe-EDDHA increased the seed Fe concentration in common bean. Moreover, the highest seed Fe concentration (65 µg g⁻¹) was observed at Elrose in 2016 compared to the rest of the environments (Table 8). The cultivars X05TH20-2 (58 µg g⁻¹) and CDC Frontier (56 µg g⁻¹) had the highest mean seed Fe concentrations compared to the rest of the cultivars, whereas CDC Vanguard had the lowest concentration (46 µg g⁻¹). The variability in Fe concentrations was mostly attributed to cultivars across locations and years. Similar findings were previously reported in chickpea [30].

The Elrose location produced a higher Fe yield compared to Moose Jaw in 2015 and 2016. The highest Fe yield (447 g ha⁻¹) was found in the AB06-156-2 cultivar at 30 kg ha⁻¹ of chelated Fe fertilizer. At the 30 kg ha⁻¹ fertilizer rate, the Fe yield of the AB06-156-2 cultivar at Elrose in 2015 increased by 12% compared to the control. The overall Fe yield increased, varying from 4 to 19% in parallel with the Fe fertilizer doses across environments (Table 9). Kumar et al. [50] showed that the application of varying levels of Fe fertilizer up to 10 kg ha⁻¹ significantly increased the Fe concentration in chickpea grain over a control. Similar findings were also reported by Sharma et al. [51], who observed that the application of chelated Fe fertilizer improved the Fe content in seeds of pigeon pea.

Locations, cultivars and their interaction significantly affected biomass. The highest biomass (228 g per five plants) was observed at Elrose in 2015 compared to the rest of the environments. These findings are similar to those reported by Kumawat et al. [52] and Sahu et al. [53], who observed that soil-applied Fe fertilizer increased biomass yield in chickpea. Similarly, in cowpea, Mahriya and Meena [54] reported that the application of Fe fertilizer improved biomass, which is consistent with our findings. Furthermore, the results are also similar to the findings of Bansal and Chahal [55], who reported that the application of 25 µg g⁻¹ Fe in mung bean grown in alkaline soil significantly increased biomass and Fe content, which is in agreement with our findings. However, previous studies conducted in chickpea and soybean reported that application of Fe-EDDHA did not result in a significant increase in biomass, which contrasts with our findings [56,57,58].

The present study also showed that ascochyta blight disease affected the yield in both locations in 2016 (Table S1). The correlation analysis showed that ascochyta blight and yield were highly correlated (r = 0.75; p ≤ 0.01) at Moose Jaw in 2016 (Table S2). Due to ascochyta blight, Fe was most likely distributed to relatively a smaller number of plants that ultimately increased the seed Fe concentration level at Moose Jaw location in 2016 compared to 2015. These findings suggested that the magnitude of the effects of ascochyta blight on seed Fe concentration depended on the cultivars and environments.

Other characteristics such as the hundred-seed weight and seed yield varied significantly (p ≤ 0.01) among locations, years, cultivars and their interactions in the two-year experiment (Table 5). For the hundred-seed weight, both locations in 2015 produced a larger seed size compared to 2016. This is mostly attributed to the ascochyta blight disease that affected the plants at both locations in 2016 (Table S1). As a result, the hundred-seed weight decreased. The highest location mean of the hundred-seed weight (36.3 g) was observed at Elrose in 2015, whereas the lowest (25.0 g) was found at Elrose in 2016 (Table S3). The highest grain yield (6904 kg ha⁻¹) was observed at Elrose in 2015, whereas the lowest (2421 kg ha⁻¹) was found at Elrose in 2016. CDC Corinne had the highest mean yield (4832 kg ha⁻¹), whereas CDC Alma had the lowest yield (2137 kg ha⁻¹) (Table S4). Mevada et al. [59] reported that the application of Fe chelates increased grain yield significantly over a control in urdbean. By applying Fe fertilizer, Bashrat et al. [34] and Goutami and Ananda [60] also found increased growth, yield and Fe content in seeds of mung bean and groundnut, respectively. A previous study in pigeon pea showed that the application of Fe fertilizer increased yield compared to a control [61]. Similar findings were also reported by Pandit et al. [39], who found that the application of Fe fertilizer to soil increased grain production and nutritional status in chickpea. These findings suggest that the mechanism for increased Fe in seeds and the improved productivity of chickpea were due to an increased supply of Fe through an enhanced Fe status in the soil that resulted in a higher Fe uptake. Kumar et al. [50] and Sahu et al. [53] reported that the application of Fe fertilizer increased the grain yield of chickpea by 17.3%. Furthermore, the hundred-seed weight and seed yield were higher in resistant cultivars such as CDC Corinne, CDC Consul, CDC Leader and CDC Frontier compared to the susceptible cultivars CDC Alma and CDC Luna. The variations in the hundred-seed weight and seed yield among cultivars could be due to the differences in their genetic constitution, physiology, and the cultivar response to various environmental conditions. Similar observations were also made by many authors in previous studies in chickpea and common bean [30,62].

The effects of Fe fertilizer on germination, node number, days to flowering, days to maturity and plant height were not significant (Table 1). However, locations, years, cultivars and their interactions significantly affected germination, node number, days to flowering, days to maturity and plant height. Germination at both locations in 2015 was higher than in 2016. For instance, germination at Elrose in 2015 was 10% and 4% higher than at Elrose and Moose Jaw in 2016 However, the node number, days to flowering, days to maturity and plant height were higher at both locations in 2016 than 2015. The variations in the above-mentioned characteristics were mostly due to ascochyta blight infestation, cultivar response to various environmental conditions, genetics and physiology. Current findings were consistent with previous studies in mung bean, chickpeas and common bean that showed that the application of Fe fertilizer did not improve vegetative growth attributes [30,50,62,63,64]. However, previous studies in cowpea, black gram and pea showed that growth characteristics were increased with the application of Fe fertilizer, which are in contrast with our findings [64,65].

5. Conclusions

Chickpea is a daily staple in many developing countries where people often affected by Fe deficiency and Fe-deficiency-related anemia. Improving the Fe concentration in the seeds of chickpea, along with increasing consumption, is one of the major strategies to correct Fe deficiency. The present study demonstrated that chickpea contained Fe at a 3.4–8.0 mg 100 g⁻¹ concentration. One of the major findings from this study is that chelated Fe fertilizer at 30 kg ha⁻¹ increased the seed Fe concentration. Results from the soil-applied Fe fertilizer show that the highest seed Fe concentration was obtained from the X05TH20-2 and CDC Frontier cultivars (58 and 56 µg Fe g⁻¹ seed, respectively). Therefore, the Fe-biofortified seeds of these two cultivars can provide approximately 6 mg Fe 100⁻¹ g seeds. Thus, 58 and 83 g servings of Fe-biofortified chickpea seeds can provide an adequate amount (50%) of Fe for children in the age groups 1–3 (7 mg Fe day⁻¹) and 4–8 (10 mg day⁻¹) years, respectively. Moreover, in the age group 19–50 years, 67 and 150 g of Fe-biofortified chickpea seeds can provide adequate amount (50%) of Fe for men (8 mg Fe day⁻¹) and women (18 mg Fe day⁻¹).

Author Contributions

T.A.J. and B.T. conceived and designed the study; T.A.J. analysed the data; T.A.J. prepared the draft manuscript; B.T., reviewed all documents critically and approved the final manuscript for submission in the journal. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The supporting data of this study are available from the corresponding author, upon request.

Acknowledgments

The authors acknowledge technical help from Jaret Horner, Scott Ife and Brent Barlow during the field trials. The authors also thank Barry Goetz for the seed iron analysis.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figure and Tables

Figure 1. The status of Fe and other macro- and micronutrients in lb ac−1 at the experimental sites at Elrose (A) and Moose Jaw (B), SK.

Figure 1. The status of Fe and other macro- and micronutrients in lb ac−1 at the experimental sites at Elrose (A) and Moose Jaw (B), SK.

Table 1

Summary of soil properties prior to fertilization and after fertilization in 2015 and 2016.

	Before Fertilization								After Fertilization
	Location								Location
	Elrose		Moose Jaw		Elrose		Moose Jaw		Elrose		Moose Jaw		Elrose		Moose Jaw
	2015				2016				2015				2016
	Depth (Inches)				Depth (Inches)				Depth (Inches)				Depth (Inches)
Soil Properties	0–6	6–12	0–6	6–12	0–6	6–12	0–6	6–12	0–6	6–12	0–6	6–12	0–6	6–12	0–6	6–12
pH	7.5	7.9	7.9	8.2	7.9	8.1	7.5	8.2	7.2	7.6	7.9	8.2	7.4	8.1	7.7	8.2
N (mg kg−1)	13.0	10.0	6.5	3.5	8.6	8.1	12.4	7.6	8.8	4.8	8.1	5.4	4.4	1.7	6.6	1.0
P (mg kg−1)	13.0	3.5	9.5	2.0	3.6	2.0	14.4	3.9	19.3	7.5	10.8	2.0	11.9	2.0	13.2	2.1
K (mg kg−1)	270	255	270	255	699	501	756	614	1120	856	849	420	932	582	885	507
Fe (mg kg−1)	20.5	11.4	13.3	9.4	19.8	19.8	18.3	17.0	22.1	23.0	16.7	13.8	20.7	21.3	22.1	19.7

Table 2

Description of eighteen cultivars used in the soil-applied Fe fertilization.

Entry	Cultivars	Types	100 Seed Weight (g)(Avg. of 2015 and 2016)
1.	1173-1	Kabuli	34.9
2.	1460-2	Desi	20.3
3.	AB06-156-2	Kabuli	37.6
4.	Amit	Kabuli	25.2
5.	CA05-75-45	Kabuli	26.5
6.	CDC Alma	Kabuli	29.3
7.	CDC Cabri	Desi	28.6
8.	CDC Consul	Desi	29.9
9.	CDC Corinne	Desi	25.5
10.	CDC Cory	Desi	26.6
11.	CDC Frontier	Kabuli	30.9
12.	CDC Leader	Kabuli	35.6
13.	CDC Luna	Kabuli	29.7
14.	CDC Orion	Kabuli	36.0
15.	CDC Palmer	Kabuli	39.3
16.	CDC Vanguard	Desi	21.9
17.	X05TH20-2	Kabuli	37.1
18.	X05TH47-3	Kabuli	35.1

Table 3

Fe (III) chelates and their stability level on high pH soil in order to correct Fe deficiency.

Fe Fertilizer	Stability	Fe Deficiency
Fe-EDTA, Fe-DTPA and Fe-HEDTA	Low	Limited/no results
Fe-EDDHA	High	Effective

Source: Adapted from [46].

Table 4

Disease rating scale (0–9) of ascochyta blight in chickpea.

Rating	Symptoms
0	No symptoms
1	Few, very small (<2 mm2) lesions on leaves and/or stems, <2% plant area affected (PAA)
2	Very small (<2 mm2) lesions, 2–5% PAA
3	Many small lesions (#2–5 mm2), 5–10% PAA
4	Many small lesions, few large (>5 mm2) lesions, 10–25% PAA
5	Many large lesions, 25–50% PAA
6	Lesions coalescing, 50–75% PAA
7	Lesions coalescing with stem girdling, 75–90% PAA
8	Stem girdling or breakage, >90% PAA
9	Plant dead

Source: Chongo et al., 2004 [47].

Table 5

Analysis of variance table showing the F values of the effects of location (LOC), year (YEAR), replication (REP), cultivar (CUL), dose (DOS) and their interactions on germination (%), node no., days to flowering, days to maturity, plant height (cm), biomass (g), 100-seed weight (g), yield (kg ha⁻¹), seed Fe (µg g⁻¹) and Fe yield (g ha⁻¹) of eighteen chickpea cultivars with three doses of Fe-EDDHA over four replications at Elrose and Moose Jaw, SK.

Sources of Variation	df	Germination	Node Number	Days to Flowering	Days to Maturity	Plant Height	Biomass	100-Seed Weight	Yield	Seed Fe Conc.
LOC	1	68.3 **	3.6 ns	1232 **	536 **	766 **	101 **	0.6 ns	1254 **	1248 **
YEAR	1	227 **	93.8 **	131 **	12804 **	5701 **	0.8 ns	1401 **	1367 **	884 **
REP	3	0.9 ns	3.0 *	3.2*	1.9 ns	4.7 *	2.4 *	8.2 **	22.0 **	12.4 **
CUL	17	4.3 **	9.9 **	12.2 **	8.2 **	17.5 **	8.1 **	117 **	35.1 **	19.3 **
DOS	2	1.3 ns	0.7 ns	1.8 ns	2.7 ns	0.3 ns	61.7 **	1.7 ns	0.0 ns	14.1 **
LOC*YEAR	1	0.9 ns	19.0 **	395 **	968 **	157 **	1292 **	99.9 **	1866 **	88.9 **
LOC*CUL	17	1.4 ns	1.4 ns	2.1 *	2.4*	1.9 *	6.0 **	3.0 **	5.9 **	2.5 *
LOC*DOS	2	1.6 ns	1.4 ns	4.3 *	15.8 **	2.0 ns	25.5 **	3.1 *	3.6 *	0.9 ns
CUL*YEAR	17	6.9 **	3.5 **	6.8 **	6.1 **	3.2 **	9.4 **	17.3 **	7.7 **	11.0 **
CUL*DOS	34	1.2 ns	1.2 ns	0.6 ns	0.7 ns	0.5 ns	1.4 ns	0.7 ns	1.6 *	0.6 ns
DOS*YEAR	2	2.5 ns	0.2 ns	1.4 ns	1.5 ns	2.6 ns	2.4 ns	1.8 ns	1.3 ns	4.1 *
LOC*YEAR*CUL	17	1.5 ns	3.1 **	6.0 **	2.4 *	2.6 *	3.2 **	4.2 **	12.6 **	2.0 *
LOC*YEAR*DOS	2	2.0 ns	0.4 ns	0.5 ns	11.3 **	0.6 ns	32.1 **	1.5 ns	1.3 ns	1.4 ns
CUL*YEAR*DOS	34	1.1 ns	1.6 *	0.7 ns	0.8 ns	0.7 ns	1.0 ns	0.8 ns	2.4 **	0.7 ns
CUL*LOC*DOS	34	0.9 ns	0.8 ns	0.6 ns	0.7 ns	0.7 ns	1.1 ns	0.7 ns	1.7 *	0.9 ns
LOC*YEAR*CUL*DOS	34	1.1 ns	1.3 ns	0.9 ns	0.8 ns	0.5 ns	1.9 *	0.8 ns	1.5 *	1.2 ns

* = significant at 5%; ** = significant at 1%; ^ns = nonsignificant; df = degrees of freedom.

Table 6

Analysis of variance and F values of the effect of location (LOC), replication (REP), cultivar (CUL), dose (DOS) and the interaction of LOC*CUL, LOC*DOS, CUL*DOS and CUL*LOC*DOS on the disease scores of eighteen chickpea cultivars with three doses of Fe-EDDHA over four replications at both sites in 2016.

Sources of Variation	df	Disease Score
LOC	1	62.4 **
REP	3	37.7 **
CUL	17	8.9 **
DOS	2	2.1 ns
LOC*CUL	17	0.6 ns
LOC*DOS	2	13.2 **
CUL*DOS	34	0.5 ns
LOC*CUL*DOS	34	0.7 ns

** = significant at 1%; ^ns = nonsignificant; df = degrees of freedom.

Table 7

The mean biomass (g plant⁻¹) of eighteen chickpea cultivars with three Fe fertilizer doses (0 kg ha⁻¹, 10 kg ha⁻¹ and 30 kg ha⁻¹) at Elrose and Moose Jaw in 2015 and 2016.

Cultivars	Elrose						Moose Jaw						Cultivar Mean
	2015			2016			2015			2016
	kg ha−1			kg ha−1			kg ha−1			kg ha−1
	0	10	30	0	10	30	0	10	30	0	10	30
1173-1	199	208	255	109	119	127	89	125	99	157	166	182	153
1460-2	175	232	244	112	117	124	118	89	105	171	258	171	159
AB06-156-2	149	280	295	101	118	146	149	127	157	198	201	208	177
Amit	195	220	244	126	136	160	118	125	148	216	246	247	182
CA05-75-45	179	198	263	125	128	149	87	108	139	173	189	263	167
CDC Alma	183	170	208	82	92	100	102	119	85	162	121	113	128
CDC Cabri	166	260	329	129	144	161	81	73	111	127	178	154	159
CDC Consul	116	215	223	133	139	143	83	126	86	176	208	261	159
CDC Corinne	167	226	260	147	148	170	130	138	134	196	217	219	179
CDC Cory	167	250	262	160	162	174	109	143	123	229	245	301	194
CDC Frontier	238	240	263	136	161	170	136	137	121	243	265	248	196
CDC Leader	195	222	235	137	141	145	94	82	86	205	216	259	168
CDC Luna	150	248	304	115	135	130	102	90	93	100	164	165	150
CDC Orion	207	316	389	130	134	148	105	109	106	191	191	197	185
CDC Palmer	166	170	215	119	132	141	113	112	95	222	229	266	165
CDC Vanguard	201	259	261	127	129	158	157	127	149	132	238	141	173
X05TH20-2	270	302	325	117	124	147	122	122	114	172	172	178	180
X05TH47-3	160	166	258	129	156	186	131	134	111	210	214	223	173
Dose Mean	182	232	269	124	134	149	112	116	115	182	206	211	169
LSD0.05	51.9	72.9	58.1	30	29.6	39.8	40.9	37.7	36.4	55.9	53.9	71.7	48.2

LSD = Least Significant Difference.

Table 8

Seed Fe concentrations (µg g⁻¹) of eighteen chickpea cultivars with three Fe fertilizer doses (0 kg ha⁻¹, 10 kg ha⁻¹ and 30 kg ha⁻¹⁾ at Elrose and Moose Jaw in 2015 and 2016.

Cultivars	Elrose						Moose Jaw						CultivarMean
	2015			2016			2015			2016
	0kg ha−1	10kg ha−1	30kg ha−1	0kg ha−1	10kg ha−1	30kg ha−1	0kg ha−1	10kg ha−1	30kg ha−1	0kg ha−1	10kg ha−1	30kg ha−1
1173-1	43.4	48.0	49.3	61.0	62.8	67.1	38.1	37.2	40.9	46.4	46.3	51.5	49.3
1460-2	47.4	52.1	54.3	56.2	59.5	61.2	38.8	39.5	43.6	45.1	44.3	49.8	49.3
AB06-156-2	50.9	52.2	57.1	67.1	70.3	71.5	41.1	42.5	41.0	46.7	49.1	54.8	53.7
Amit	48.7	50.0	48.0	59.0	64.8	71.8	40.4	41.5	43.1	46.0	48.8	50.5	51.1
CA05-75-45	48.0	42.5	48.8	57.8	56.7	59.0	34.9	34.1	34.3	48.9	44.4	50.7	46.7
CDC Alma	43.4	50.1	49.6	64.9	71.8	79.9	37.7	39.1	38.5	48.4	50.3	53.9	52.3
CDC Cabri	45.5	46.4	53.8	57.2	57.4	59.6	37.2	39.9	39.6	47.3	47.5	51.2	48.5
CDC Consul	52.1	51.6	53.5	56.9	62.8	57.4	41.8	47.1	43.0	40.7	44.4	47.4	49.9
CDC Corinne	45.7	49.7	50.1	55.1	54.6	60.4	40.6	39.4	40.6	40.8	40.8	42.8	46.7
CDC Cory	46.9	48.4	51.2	56.0	59.5	64.3	42.3	43.9	44.9	42.1	44.9	47.8	49.3
CDC Frontier	57.0	54.6	55.3	66.3	72.7	75.6	43.3	44.5	46.3	49.2	51.6	56.9	56.1
CDC Leader	48.2	47.2	45.3	60.3	60.0	62.1	37.6	37.0	39.2	43.1	45.7	52.7	48.2
CDC Luna	47.6	47.1	48.4	78.0	73.3	80.0	37.8	38.7	37.2	48.7	56.0	55.5	54.0
CDC Orion	45.7	53.4	52.2	69.6	68.1	71.0	39.4	37.1	39.7	46.7	51.9	54.4	52.4
CDC Palmer	48.4	48.4	48.5	64.1	63.3	68.8	36.5	40.1	39.4	45.6	44.5	47.8	49.6
CDC Vanguard	43.5	47.5	47.8	52.9	48.8	56.5	40.3	39.4	47.1	41.0	44.0	48.4	46.4
X05TH20-2	55.2	57.4	57.9	75.0	72.1	75.6	48.5	50.5	46.1	50.3	51.4	53.6	57.8
X05TH47-3	52.6	49.5	55.5	71.9	67.3	70.0	45.7	48.9	48.3	48.6	52.3	51.4	55.2
Dose Mean	48.3	49.8	51.5	62.7	63.7	67.3	40.1	41.1	41.8	45.9	47.7	51.2	50.9
LSD0.05	7.0	11.4	7.6	9.6	11.6	9.3	4.9	4.8	5.5	7.1	5.5	6.8	7.6

LSD = Least Significant Difference.

Table 9

The mean Fe yields (g ha⁻¹) of eighteen chickpea cultivars with three different doses (0 kg ha⁻¹, 10 kg ha⁻¹ and 30 kg ha⁻¹) of Fe-EDDHA at Elrose and Moose Jaw in 2015 and 2016.

Cultivars	Elrose						Moose Jaw						CultivarMean
	2015			2016			2015			2016
	kg ha−1			kg ha−1			kg ha−1			kg ha−1
	0	10	30	0	10	30	0	10	30	0	10	30
1173-1	311	347	316	129	116	31	86	83	111	141	131	170	164
1460-2	340	337	370	137	125	126	113	91	113	171	132	184	187
AB06-156-2	396	410	447	137	171	125	93	104	104	145	98	199	202
Amit	306	337	335	132	113	174	93	86	90	132	150	150	175
CA05-75-45	305	250	311	160	132	138	74	59	61	112	171	126	158
CDC Alma	234	250	247	62	65	63	89	79	109	.	.	.	133
CDC Cabri	291	326	319	284	172	188	74	99	90	152	72	107	181
CDC Consul	430	405	419	172	171	135	132	127	129	196	253	291	238
CDC Corinne	405	438	434	174	248	211	122	110	100	149	182	189	230
CDC Cory	376	351	374	262	264	155	122	144	159	127	203	141	223
CDC Frontier	443	433	428	130	150	314	140	131	131	78	62	75	210
CDC Leader	359	420	367	152	135	69	98	104	104	190	156	165	193
CDC Luna	282	294	291	149	112	141	100	107	110	14	8.0	30	137
CDC Orion	267	379	315	140	96	185	106	96	104	106	89	81	164
CDC Palmer	416	342	422	190	206	204	98	96	98	173	170	300	226
CDC Vanguard	272	372	288	168	151	156	105	95	94	108	97	104	167
X05TH20-2	263	334	306	109	97	147	85	92	83	104	98	109	152
X05TH47-3	346	367	387	133	182	113	103	141	123	198	153	209	205
Dose Mean	336	355	354	157	150	149	102	102	106	135	131	155	186
LSD0.05	73.4	90.8	69.6	73	98.8	136.2	24.8	24.5	34.4	57.3	59.6	65.7	67.3

LSD = Least Significant Difference.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy13122894/s1, Table S1: Disease score (0–9) of eighteen chickpea cultivars with three different doses of Fe-EDDHA at Elrose and Moose Jaw in 2016. Table S2. Correlation between ascochyta blight score and yield as well as Fe yield of eighteen chickpea cultivars with three different doses of Fe-EDDHA at Elrose and Moose Jaw in 2016. Table S3. Hundred seed weight (g) of eighteen chickpea cultivars with three different doses of Fe-EDDHA at Elrose and Moose Jaw in the year 2015 and 2016. Table S4. Average yield (kg ha⁻¹) of eighteen chickpea cultivars across all doses of Fe-EDDHA at Elrose and Moose Jaw in the year 2015 and 2016.

References

1. Grillet, L.; Mari, S.; Schmidt, W. Fe in seeds—Loading pathways and subcellular localization. Front. Plant Sci.; 2014; 4, 535. [DOI: https://dx.doi.org/10.3389/fpls.2013.00535]

2. Tan, G.Z.H.; Das Bhowmik, S.S.; Hoang, T.M.L.; Karbaschi, M.R.; Johnson, A.A.T.; Williams, B.; Mundree, S.G. Finger on the pulse: Pumping Fe into chickpea. Front. Plant Sci.; 2017; 8, 1755. [DOI: https://dx.doi.org/10.3389/fpls.2017.01755]

3. Zuo, Y.; Zhang, F. Soil and crop management strategies to prevent Fe deficiency in crops. Plant Soil; 2011; 339, pp. 83-95. [DOI: https://dx.doi.org/10.1007/s11104-010-0566-0]

4. Colombo, C.; Palumbo, G.; He, J.Z.; Pinton, R.; Cesco, S. Review on Fe availability in soil: Interaction of Fe minerals, plants, and microbes. J. Soils Sediments; 2014; 14, pp. 538-548. [DOI: https://dx.doi.org/10.1007/s11368-013-0814-z]

5. Boukhalfa, H.; Crumbliss, A.L. Chemical aspects of siderophore mediated Fe transport. Biometals; 2002; 15, pp. 325-339. [DOI: https://dx.doi.org/10.1023/A:1020218608266]

6. Römheld, V.; Marschner, H. Evidence for a specific uptake system for Fe phytosiderophores in roots of grasses. Plant Physiol.; 1986; 80, pp. 175-180. [DOI: https://dx.doi.org/10.1104/pp.80.1.175]

7. Robinson, N.J.; Procter, C.M.; Connolly, E.L.; Guerinot, M.L. A ferric-chelate reductase for Fe uptake from soils. Nature; 1999; 397, pp. 694-697. [DOI: https://dx.doi.org/10.1038/17800]

8. Morrissey, J.; Guerinot, M. Fe uptake and transport in plants: The good, the bad, and the ionome. Chem. Rev.; 2009; 109, pp. 4553-4567. [DOI: https://dx.doi.org/10.1021/cr900112r]

9. Jeong, J.; Connolly, E.L. Fe uptake mechanisms in plants: Functions of the FRO family of ferric reductases. Plant Sci.; 2009; 176, pp. 709-714. [DOI: https://dx.doi.org/10.1016/j.plantsci.2009.02.011]

10. Chugh, V.; Dhaliwal, H. Biofortification of Staple Crops. Agricultural Sustainability; Elsevier: Amsterdam, The Netherlands, 2013; pp. 177-196. [DOI: https://dx.doi.org/10.1016/B978-0-12-404560-6.00009-5]

11. Mayer, J.; Pfeiffer, W.; Beyer, P. Biofortified crops to alleviate micronutrient malnutrition. Curr. Opin. Plant Biol.; 2008; 11, pp. 166-170. [DOI: https://dx.doi.org/10.1016/j.pbi.2008.01.007]

12. Briat, J.F. Fe Nutrition and Implications for Biomass Production and the Nutritional Quality of Plant Products. Molecular and Physiological Basis of Nutrient Use Efficiency in Crops; Hawkesford, M.J.; Barraclough, P. Wiley-Blackwell: Oxford, UK, 2011; pp. 309-328.

13. Cakmak, I. Enrichment of cereal grains with zinc: Agronomic or genetic biofortification?. Plant Soil; 2008; 302, pp. 1-17. [DOI: https://dx.doi.org/10.1007/s11104-007-9466-3]

14. De Valença, A.; Bake, A.; Brouwer, I.; Giller, K. Agronomic biofortification of crops to fight hidden hunger in sub-Saharan Africa. Glob. Food Secur.; 2017; 12, pp. 8-14. [DOI: https://dx.doi.org/10.1016/j.gfs.2016.12.001]

15. Prasad, R. Ferti-fortifcation of grains an easy option to alleviate malnutrition of some micronutrients in human beings. Indian J. Fertil.; 2009; 5, pp. 129-133.

16. Manzeke, G.; Mapfumo, M.; Mtambanengwe, P.; Chikowo, F.; Tendayi, R.; Cakmak, T. Soil fertility management effects on maize productivity and grain zinc content in smallholder farming systems of Zimbabwe. Plant Soil; 2012; 361, pp. 57-69. [DOI: https://dx.doi.org/10.1007/s11104-012-1332-2]

17. Vanlauwe, B.; Descheemaeker, K.; Giller, K.; Huising, J.; Merckx, R.; Nziguheba, G.; Wendt, J.; Zingore, S. Integrated soil fertility management in sub-Saharan Africa: Unravelling local adaptation. Soil; 2015; 1, pp. 491-508. [DOI: https://dx.doi.org/10.5194/soil-1-491-2015]

18. Voortman, R.L.; Bindraban, P.S. Beyond N and P: Towards a Land Resource Ecology Perspective and Impactful Fertilizer Interventions in Sub-Sahara Africa; VFRC Report 2015/1 Virtual Fertilizer Research Center: Washington, DC, USA, 2015; 49.

19. Singh, M.; Prasad, K. Agronomic Aspects of Zinc Biofortification in Rice (Oryza sativa L.). Proc. Natl. Acad. Sci. India Sect. B Biol. Sci.; 2014; 84, pp. 613-623. [DOI: https://dx.doi.org/10.1007/s40011-014-0329-4]

20. Rietra, R.P.J.J.; Heinen, M.; Dimpla, C.; Bindraban, P.S. Effects of Nutrients Antagonism and Synergism on Fertilizer Use Efficiency; VFRC Report 2015/5 Virtual Fertilizer Research Center: Washington, DC, USA, 2015; Available online: http://www.vfrc.org/getdoc/e738b7d3-8f70-4b18-b3d9-980694b5f26c/vfrc_2015-5_effects_of_nutrient_antagonism_and_syn.pdf (accessed on 20 January 2018).

21. Walworth, D.J. Recognizing and Treating Fe Deficiency in the Home Yard. Available online: https://extension.arizona.edu/sites/extension.arizona.edu/files/pubs/az1415.pdf (accessed on 10 May 2019).

22. Millán, T.; Madrid, E.; Cubero, J.I.; Amri, M.; Patricia, C.; Rubio, J. Chickpea. Handbook of Plant Breeding; De Ron, A.M.D. Springer: Pontevedra, Spain, 2015; pp. 85-88. [DOI: https://dx.doi.org/10.1007/978-1-4939-2797-5]

23. Zhu, H.; Choi, H.; Cook, D.R.; Shoemaker, R.C. Bridging model and crop legumes through comparative genomics. Plant Physiol.; 2005; 137, pp. 1189-1196. [DOI: https://dx.doi.org/10.1104/pp.104.058891]

24. FAOSTAT. FAO Statistical Database. Available online: http://www.fao.org/faostat/en/#data/QC (accessed on 15 May 2018).

25. Akibode, C.S.; Maredia, M.K. Global and Regional Trends in Production, Trade and Consumption of Food Legume Crops; Michigan State University: East Lansing, MI, USA, 2012; [DOI: https://dx.doi.org/10.22004/ag.econ.136293]

26. Wells, H.F.; Bond, J.K. Vegetables and Pulses Yearbook Data. Economic Research Service, USDA. 2016; Available online: https://downloads.usda.library.cornell.edu/usda-esmis/files/1n79h429p/z890rw81t/rj430695g/VGS-08-30-2016.pdf (accessed on 15 March 2019).

27. Ibrikci, H.; Knewtson, S.; Grusak, M. Chickpea leaves as a vegetable green for humans: Evaluation of mineral composition. J. Sci. Food Agric.; 2003; 83, pp. 945-950. [DOI: https://dx.doi.org/10.1002/jsfa.1427]

28. Yadav, S.S.; Longnecker, N.; Dusunceli, F.; Bejiga, G.; Yadav, M.; Rizvi, A.H.; Manohar, M.; Reddy, A.A.; Xaxiao, Z.; Chen, W. Uses, consumption and utilization. Chickpea Breeding and Management; Yadav, S.S.; Redden, R.J.; Chen, W.; Sharma, B. CABI: Cambridge, MA, USA, 2007; pp. 72-100. [DOI: https://dx.doi.org/10.2139/ssrn.1735326]

29. Diapari, M.; Sindhu, A.; Bett, K.; Deokar, A.; Warkentin, T.; Tar’an, B. Genetic diversity and association mapping of Fe and zinc concentrations in chickpea (Cicer arietinum L.). Genome; 2014; 57, pp. 459-468. [DOI: https://dx.doi.org/10.1139/gen-2014-0108]

30. USDA. National Nutrient Database for Standard Reference. Available online: http://www.ars.usda.gov/Services?docs.htm?docid=8964 (accessed on 25 March 2018).

31. Chilimba, A.; Young, S.; Black, C.; Meacham, M.; Lammel, J.; Broadley, M. Agronomic biofortification of maize with selenium (Se) in Malawi. Field Crops Res.; 2012; 125, pp. 118-128. [DOI: https://dx.doi.org/10.1016/j.fcr.2011.08.014]

32. Yilmaz, A.; Ekiz, H.; Torun, B.; Gultekin, I.; Karanlik, S.; Bagci, S.A.; Cakmak, I. Effect of different zinc application methods on grain yield and zinc concentration in wheat cultivars grown on zinc-deficient calcareous soils. J. Plant. Nutr.; 1997; 20, pp. 461-471. [DOI: https://dx.doi.org/10.1080/01904169709365267]

33. Hidoto, L.; Worku, W.; Mohammed, H.; Bunyamin, T. Effects of zinc application strategy on zinc content and productivity of chickpea grown under zinc deficient soils. J. Soil Sci. Plant Nutr.; 2017; 17, pp. 112-126. [DOI: https://dx.doi.org/10.4067/S0718-95162017005000009]

34. Ali, B.; Ali, A.; Tahir, M.; Ali, S. Growth, Seed yield and quality of mungbean as influenced by foliar application of Fe sulfate. Pak. J. Life Soc. Sci.; 2014; 12, pp. 20-25. Available online: https://pjlss.edu.pk/pdf_files/2014_1/4)%20Ali%20et%20al%202014%20(1).pdf (accessed on 25 March 2018).

35. Smrkolj, P.; Germ, M.; Kreft, I.; Stibilj, V. Respiratory potential and Se compounds in pea (Pisum sativum L.) plants grown from Se-enriched seeds. J. Exp. Bot.; 2006; 57, pp. 3595-3600. [DOI: https://dx.doi.org/10.1093/jxb/erl109]

36. Smrkolj, P.; Osvald, M.; Osvald, J.; Stibilj, V. Selenium uptake and species distribution in selenium-enriched bean (Phaseolus vulgaris L.) seeds obtained by two different cultivations. Eur. Food Res. Technol.; 2007; 225, pp. 233-237. [DOI: https://dx.doi.org/10.1007/s00217-006-0409-7]

37. Molina, M.G.; Quiroz, C.M.; de la Cruz, L.E.; Martinez, J.R.V.; Parra, J.M.S.; Carrillo, M.G.; Vidal, J.A.O. Biofortification of cowpea beans (Vigna unguiculata L. Walp) with Fe and zinc. Mex. J. Agric. Sci.; 2016; 17, pp. 3427-3438.

38. Sida-Arreola, J.; Sánchez, E.; Ojeda-Barrios, D.; Ávila-uezada, G.; Flores-Córdova, M.; Márquez-Quiroz, C.; Preciado-Rangel, P. Can biofortification of zinc improve the antioxidant capacity and nutritional quality of beans?. Emir. J. Food Agric.; 2017; 29, pp. 237-241.

39. Rathod, S.P.; Patil, D.H.; Bellad, S.B.; Haveri, V.R. Biofortification of Zn and Fe in Chickpea through Agronomic Intervention in Medium Black Soils of Karnataka. Legume Res.; 2022; 45, pp. 981-987. [DOI: https://dx.doi.org/10.18805/LR-4341]

40. Márquez-Quiroz, C.; De-La-Cruz-Lázaro, E.; Osorio-Osorio, R.; Sánchez-Chávez, E. Biofortification of cowpea beans with Fe: Fe’s influence on mineral content and yield. J. Soil Sci. Plant Nutr.; 2015; 15, pp. 839-847.

41. Hussain, S.; Rengel, Z.; Aziz, T.; Abid, M. Estimated Zinc Bioavailability in Milling Fractions of Biofortified Wheat Grains and in Flours of Different Extraction Rates. Int. J. Agric. Biol.; 2013; 15, pp. 921-926.

42. White, P.J.; Broadley, M.R. Biofortification of crops with seven mineral elements often lacking in human diets. New Phytol.; 2009; 182, pp. 49-84. [DOI: https://dx.doi.org/10.1111/j.1469-8137.2008.02738.x]

43. Alfthan, G.; Eurola, M.; Ekholm, P.; Venäläinen, E.; Root, T.; Korkalainen, K.; Hartikainen, H.; Salminen, P.; Hietaniemi, V.; Aspila, P. et al. Effects of nationwide addition of selenium to fertilizers on foods, and animal and human health in Finland: From deficiency to optimal selenium status of the population. J. Trace Elem. Med. Biol.; 2015; 31, pp. 142-147. [DOI: https://dx.doi.org/10.1016/j.jtemb.2014.04.009]

44. Jahan, T.A.; Vandenberg, A.; Glahn, R.P.; Tyler, R.T.; Reaney, M.J.T.; Tar’an, B. Iron Fortification and Bioavailability of Chickpea (Cicer arietinum L.) Seeds and Flour. Nutrients; 2019; 11, 2240. [DOI: https://dx.doi.org/10.3390/nu11092240]

45. Seleiman, M.; Abdelaal, M. Effect of Organic, Inorganic and Bio-fertilization on Growth, Yield and Quality Traits of Some Chickpea (Cicer arietinum L.) Varieties. Egypt. J. Agron.; 2018; 40, pp. 105-117. [DOI: https://dx.doi.org/10.21608/agro.2018.2869.1093]

46. Cropnuts. Available online: https://cropnuts.helpscoutdocs.com/article/826-iron-fertilisation (accessed on 15 September 2020).

47. Chongo, G.; Gossen, B.D.; Buchwaldt, L.; Adhikari, T.; Rimmer, S.R. Genetic diversity of Ascochyta rabiei in Canada. Plant Dis.; 2004; 88, pp. 4-10. [DOI: https://dx.doi.org/10.1094/PDIS.2004.88.1.4]

48. DellaValle, D.M.; Thavarajah, D.; Thavarajah, P.; Vandenberg, A.; Glahn, R.P. Lentil (Lens culinaris L.) as a candidate crop for Fe biofortification: Is there genetic potential for Fe bioavailability?. Field Crops Res.; 2013; 144, pp. 119-125. [DOI: https://dx.doi.org/10.1016/j.fcr.2013.01.002]

49. Moraghan, J.; Padilla, T.; Etchevers, J.; Grafton, K.; Acosta-Gallegos, J. Fe accumulation in seed of common bean. Plant Soil; 2002; 246, pp. 175-183. [DOI: https://dx.doi.org/10.1023/A:1020616026728]

50. Kumar, V.; Dwivedi, V.N.; Tiwari, D.D. Effect of phosphorous and Fe on yield and mineral nutrition in chickpea. Ann. Plant Soil Res.; 2009; 11, pp. 16-18.

51. Sharma, S.; Sharma, M.; Ramesh, A. Biofortification of crops with micronutrients through agricultural approaches. Indian Farming; 2010; 60, pp. 7-12.

52. Kumawat, R.N.; Rathore, P.S.; Pareek, N. Response of mung bean to sulphur and Fe nutrition grown on calcareous soil of Western Rajasthan. Indian Soc. Pulses Res. Dev.; 2006; 19, pp. 228-230.

53. Sahu, S.; Lidder, R.S.; Singh, P.K. Effect of micronutrients and biofertilizers on growth, yield and nutrient uptake by chickpea (Cicer aeritinum L.) in Vertisols of Madhya Pradesh. Adv. Plant Sci.; 2008; 21, pp. 501-503.

54. Mahriya, A.K.; Meena, N. Response of phosphorous and Fe on growth and quality of cowpea (Vigna unguiculata L.). Ann. Agri-Bio Res.; 1999; 4, pp. 203-205.

55. Bansal, R.L.; Chahal, D.S. Interaction effect of Fe and Mn on growth and nutrient content of moong (Phaseolus aureus L.). Acta Agron. Hung.; 1990; 39, pp. 59-63.

56. Ghasemi-Fasaei, R.; Ronaghi, A.; Maftoun, M.; Karimian, N.; Soltanpour, P. Fe-Manganese Interaction in Chickpea as Affected by Foliar and Soil Application of Fe in a Calcareous Soil. Commun. Soil Sci. Plant Anal.; 2005; 36, pp. 1717-1725. [DOI: https://dx.doi.org/10.1081/CSS-200062428]

57. Moosavi, A.A.; Ronaghi, A. Influence of foliar and soil applications of Fe and manganese on soybean dry matter yield and Fe-manganese relationship in a calcareous soil. Aust. J. Crop Sci.; 2011; 5, pp. 1550-1556.

58. Ronaghi, A.; Ghasemi-Fasaei, R. Field Evaluations of Yield, Fe-Manganese Relationship, and Chlorophyll Meter Readings in Soybean Genotypes as Affected by Fe-Ethylenediamine Di-o-hydroxyphenylacetic Acid in a Calcareous Soil. J. Plant Nutr.; 2007; 31, pp. 81-89. [DOI: https://dx.doi.org/10.1080/01904160701741925]

59. Mevada, K.D.; Patel, J.J.; Patel, K.P. Effect of micronutrients on yield of urdbean. Indian Soc. Pulses Res. Dev.; 2005; 18, pp. 214-216.

60. Gowthami, S.S.; Ananda, N. Effect of zinc and iron ferti-fortification on growth, pod yield and zinc uptake of groundnut (Arachis hypogaea L.) genotypes. Int. J. Agric. Environ. Biotechnol.; 2015; 10, pp. 575-580. [DOI: https://dx.doi.org/10.5958/2230-732X.2017.00070.5]

61. Hanumanthappa, D.; Vasudevan,; Maruthi, S.; Shakuntala, J.B.; Muniswamy, N.M.; Macha, S.I. Enrichment of iron and zinc content in pigeonpea genotypes through agronomic biofortification to mitigate malnutrition. Int. J. Curr. Microbiol. Appl. Sci.; 2018; 7, pp. 4334-4342.

62. Ariza-Nieto, M.; Blair, M.; Welch, R.; Glahn, R. Screening of Fe bioavailability patterns in eight bean (Phaseolus vulgaris L.) genotypes using the Caco-2 cell in vitro model. J. Agric. Food Chem.; 2007; 55, pp. 7950-7956. [DOI: https://dx.doi.org/10.1021/jf070023y]

63. Janmohammadi, M.; Abdoli, H.; Sabaghnia, N.; Esmailpour, M.; Aghaei, A. The Effect Of Fe, Zinc and Organic Fertilizer on Yield of Chickpea (Cicer artietinum L.) in Mediterranean Climate. Acta Univ. Agric. Silvic. Mendel. Brun.; 2018; 66, pp. 0049-0060. [DOI: https://dx.doi.org/10.11118/actaun201866010049]

64. Shukla, V.S.I. Effect of Fe, Mo, Zn and P on symbiotic nitrogen fixation of chickpea. Indian J. Agric. Chem.; 1994; 32, pp. 118-123.

65. Thapu, U.; Rai, P.; Suresh, C.P.; Pal, P. Effect of micronutrients on the growth and yield of pea in gangetic alluvial of West Bengal. Environ. Ecol.; 2003; 21, pp. 179-182.